108 MICROSCOPIC METHODS 



made up of equal parts of saturated alcoholic solution of crystal -violet 

 (" Krystall-violet ") and 1 per cent, solution of ammonium carbonate. 



(2) Wash in water. 



(3) Place for two or three minutes in Gram's iodine solution, or in 

 the following modification by Kiihne : 



Iodine ...... 2 parts. 



Potassium iodide . . . . 4 ,, 



Distilled water 100 ,, 



For use, dilute with water to make a sherry-coloured solution. 



(4) Wash in water. 



(5) Decolorise in a saturated alcoholic solution of fluorescein (a 

 saturated solution in methylated spirit does equally well). 



(6) Dehydrate, clear, and mount. 



There is great variability in the avidity with which organ- 

 isms stained by Gram retain the dye when washed with alcohol, 

 and sometimes difficulty is experienced in saying whether 

 an organism does or does not stain by this method. Most 

 bacteria are either frankly Gram-positive or frankly Gram- 

 negative, but cases occur where an organism, usually Gram- 

 positive or Gram- negative, tends when grown on certain media to 

 show an opposite tendency, and sometimes an organism is met 

 with in which the individuals in a film show slightly different 

 reactions to the Gram stains. According to Unna, the Gram- 

 stain can only be carried out with the pararosanilin group of 

 dyes (e.g., victoria blue, methyl violet, crystal violet, or gentian 

 violet, which is a mixture of the last two). Two theories, a 

 chemical and a physical, have been advanced to explain the 

 reaction. According to the former, the iodine combines with 

 the dye and links it to the bacterial protoplasm. According to 

 the physical, the stain is deposited in the protoplasm, and is 

 relatively slowly washed out by alcohol. The iodine penetrates 

 Gram-positive bacteria most readily and causes a more pronounced 

 deposit of the stain. Recent work indicates that the physical 

 explanation is the more probable, and that differences in the 

 capsule of the two classes of bacteria is an important factor in 

 the reaction. 



Stain for Tubercle and other Acid-fast Bacilli. These 

 bacilli cannot be well stained with a simple watery solution of 

 a basic aniline dye. This fact can easily be tested by attempt- 

 ing to stain a film of a tubercle culture with such a solution ; 

 with the Gram method, however, a partial staining is effected. 

 Such bacteria require a powerful stain containing a mordant, 

 and must be exposed to the stain for a long time, or its action 



